Structural basis of the transcription termination factor Rho engagement with transcribing RNA polymerase from Thermus thermophilus

Transcription termination is an essential step in transcription by RNA polymerase (RNAP) and crucial for gene regulation. For many bacterial genes, transcription termination is mediated by the adenosine triphosphate–dependent RNA translocase/helicase Rho, which causes RNA/DNA dissociation from the RNAP elongation complex (EC). However, the structural basis of the interplay between Rho and RNAP remains obscure. Here, we report the cryo–electron microscopy structure of the Thermus thermophilus RNAP EC engaged with Rho. The Rho hexamer binds RNAP through the carboxyl-terminal domains, which surround the RNA exit site of RNAP, directing the nascent RNA seamlessly from the RNA exit to its central channel. The β-flap tip at the RNA exit is critical for the Rho-dependent RNA release, and its deletion causes an alternative Rho-RNAP binding mode, which is irrelevant to termination. The Rho binding site overlaps with the binding sites of other macromolecules, such as ribosomes, providing a general basis of gene regulation.

(A) A schematic representation of the DNA and RNA for EC preparation. Sequences of the sense (non-template) strand of DNA variants and the primer locations are shown.
(B) NusG and GreA do not bind tightly to the EC. The PstI-digested template DNA (2 pmol) was mixed with 10 µl of magnetic bead solution in buffer A. After an incubation for 15 minutes at room temperature, the beads were washed three times with M buffer. Afterwards, 2 pmol of RNAP, 4 pmol of GreA, 4 pmol of NusG, and 2 nmol of substrates ATP/UTP/CTP were added to the beads in 40 µl of M buffer, and the mixture was incubated for 15 minutes at 65˚C. Aliquots (10 µl) of the supernatant fraction and the entire bead fraction after three washes with M buffer were analyzed by SDS-PAGE.
(C) RNA release assay with NusG. The bead-immobilized EC was prepared without NusG. NusG (2 pmol per sample) or buffer was added to the EC and incubated at room temperature for 5 minutes, and then a mixture of Rho and ATP was added and incubated at 35°C. Mean values of three independent experiments were plotted (error bars = S.D.) (Table S2).
(D) RNA release assay with GreA. The bead-immobilized EC was prepared without GreA. GreA (2 pmol per sample) or buffer was added to the EC and incubated at room temperature for 5 minutes, and then a mixture of Rho and ATP was added and incubated at 35°C. Mean values of three independent experiments were plotted (error bars = S.D.) (Table S2). (A) Electrophoretic analyses of gel filtration fractions: proteins were detected with SDS-PAGE and DNA/RNA were detected with Urea-PAGE. Center and right are identical Urea-PAGE gels: after detection of fluorescent RNA (center), DNA and RNA were detected by staining gels with SYBR Gold (right). Fractions marked with red boxes were collected for cryo-EM sample preparation.
(B) A raw micrograph of a cryo-EM grid used for this study.
(C) 2D class averages for the RNAP and RNAP-Rho complex particles.
(D) 2D class averages for the Rho-hexamer particles.
(E) A close-up view of ADP·BeF3 bound between Rho protomers A and B. The ADP is shown in a stick model, and the beryllium and fluoride atoms are shown as spheres. The density map is shown as mesh colored to match the model.

Fig. S3. Workflow of the image analysis.
Workflow of single-particle image analysis. Further analyses of particles containing RNAP are shown in Fig. S4. (B) Image analysis of the type-2 complex. (C) Image analysis of EC and apo RNAP.
(D) Comparison of RNAP conformations. Left: RNAP in the type-1 complex is superimposed with that in the EC by the RNAP core module (the two a subunits, residues 1-17, 394-700, 833-997 of the b subunit, and residues 781-1069 of the bʹ subunit). Right: RNAP in the type-2 complex is superimposed with apo RNAP and EC by the RNAP core module. (E) Conformational change in the w subunit C-terminal helix in the type-1 complex. The structure of the Rho-unbound EC (Figs. 1D, S4C) is superimposed on the type-1 complex, and part of the w C-helix (residues w81-96) is shown. While the type-1 complex is colored as in Fig.  2C, the w C-helix part of the Rho-unbound EC is colored moss green.